Growth and Break-Up of Methanogenic Granules Suggests Mechanisms for Biofilm and Community Development

Abstract

Methanogenic sludge granules are densely packed, small, spherical biofilms found in anaerobic digesters used to treat industrial wastewaters, where they underpin efficient organic waste conversion and biogas production. Each granule theoretically houses representative microorganisms from all of the trophic groups implicated in the successive and interdependent reactions of the anaerobic digestion (AD) process. Information on exactly how methanogenic granules develop, and their eventual fate will be important for precision management of environmental biotechnologies. Granules from a full-scale bioreactor were size-separated into small (0.6-1 mm), medium (1-1.4 mm), and large (1.4-1.8 mm) size fractions. Twelve laboratory-scale bioreactors were operated using either small, medium, or large granules, or unfractionated sludge. After >50 days of operation, the granule size distribution in each of the small, medium, and large bioreactor sets had diversified beyond-to both bigger and smaller than-the size fraction used for inoculation. Interestingly, extra-small (XS; <0.6 mm) granules were observed, and retained in all of the bioreactors, suggesting the continuous nature of granulation, and/or the breakage of larger granules into XS bits. Moreover, evidence suggested that even granules with small diameters could break. "New" granules from each emerging size were analyzed by studying community structure based on high-throughput 16S rRNA gene sequencing. Methanobacterium, Aminobacterium, Propionibacteriaceae, and Desulfovibrio represented the majority of the community in new granules. H2-using, and not acetoclastic, methanogens appeared more important, and were associated with abundant syntrophic bacteria. Multivariate integration (MINT) analyses identified distinct discriminant taxa responsible for shaping the microbial communities in different-sized granules.

Keywords: anaerobic digestion; biofilms; methanogens; microbial communities; sludge granules; wastewater.

FIGURE 1

Schematics illustrating: (a) the AD pathway of organic matter degradation in the context of a granule; (b) theoretical distribution of the main trophic groups catalyzing the process; (c) the engineered bioreactor system used to apply granules for wastewater treatment and biogas generation; (d) size distribution of biomass whereby granules were binned for this study into five size groups: extra-small (XS), small (S), medium (M), large (L), and extra-large (XL); and (e) the experimental set-up used to test granular growth where bioreactors were inoculated with either S, M, L, or the naturally distributed (mixed) biomass.

FIGURE 2

Methane yield efficiency; COD conversions (n = 3); and key VFA (acetate, propionate, and butyrate) contributions to effluent sCOD; in each of the four bioreactor sets: (A) R_S1–R_S3; (B) R_M1–R_M3; (C) R_L1–R_L3; (D) R_N1–R_N3.

FIGURE 3

Changes in distribution of granule sizes in the R_S, R_M, R_L, and R_N bioreactors during the trial (day 0 and each of the respective bioreactors at day 51), showing: (A) R_S1–R_S3; (B) R_M1–R_M3; (C) R_L1–R_L3; (D) R_N1 and R_N3 bioreactors. Colors indicate the granule size classification and their proportion of the total biomass present. (E) Map indicating frequency of observations of emerging sizes across the experiment. No sequencing data available for samples marked with (*).

FIGURE 4

Box plots (A–D) of rarefied richness of the various size classifications from across the four bioreactor sets: (A) R_S1–R_S3; (B) R_M1–R_M3; (C) R_L1–R_L3; (D) R_N1 and R_N3; and bar chart (E) showing the top 25 relatively most abundant OTUs in original and new granules. Lines for figures (A–D) connect samples where differences were significant (ANOVA) indicated by *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Granular growth and biofilm development model. (a) Operation of the model inside an anaerobic bioreactor; (b) size fraction parameters; and (c) the generalized growth model including the break-up of larger granules to form new, smaller granules.